The present invention generally concerns handling of embryos. The invention also concerns handling of oocytes (prefertilized embryos), and eggs. Embryo, as used herein, therefore encompasses oocytes, and eggs as well as fertilized embryos. The invention more specifically concerns microfluidic handling of embryos for culturing, manipulation, and analysis.
Technology assisted reproduction techniques in which embryos are handled independently from their mammalian biological source are growing in importance and frequency of use. Such techniques have great direct benefit to persons unable to have babies through unassisted sexual reproduction. The agricultural industries also increasingly rely upon such assisted reproduction techniques. Embryo manipulation is used in livestock reproduction to control such things as the faster genetic evolution of cattle and permitting the genetic characteristics of a single exceptional cow or bull to be passed on to far greater numbers of offspring than would be possible through unassisted sexual reproduction.
Livestock embryo manipulation is becoming more routine due to the development of gene manipulation, cloning, and in vitro fertilization (IVF) techniques. The overall goal of embryo manipulation in livestock is to increase production efficiency, especially with regard to reproduction, milk production or production of specific milk components, lean tissue growth with reduced fat content and decreased susceptibility to specific diseases. Embryo transfer is also used to introduce or rescue valuable germplasm and propagate rare breeding animals such as endangered exotic species.
Expense and relatively low success rates place significant burdens on the use of these assisted reproduction techniques for humans as well as livestock. In human reproduction such expense and failure adds emotional as well as economic burdens. In addition, safeguards against failures often result in unwanted or unmanageable multiple births, as well as additional stored embryos which require maintenance and additional difficult decision making at some later point in time. Expense is the primary concern in livestock reproduction.
Failure rates in reproduction techniques as well as testing and other embryo handling techniques are attributable primarily to the significant handling and manipulation of embryos in executing these techniques. Animal reproductive technologies have advanced in recent years, but the physical tools used in animal reproduction have not changed significantly. Fine-bore glass pipets are still one of the basic tools of the embryologist. Using standard petri dishes, procedures such as in vitro maturation of eggs (IVM), in vitro fertilization, and embryo culture (EC) require picking up and placing individual eggs and embryos several times for each procedure.
Such handling and movement from one petri dish to another provides significant potential for damage or contamination. Perhaps more important, though, is the failure of a stationary embryo in a petri dish to simulate the corresponding natural biological reproduction condition. Some efforts have been made to move embryos in petri dishes via agitation of the dish, but this is a haphazard approach. Expense is also created here due to the relatively large amount of biological medium required for the manual petri dish conventional embryo handling methods. Bovine embryos are individually handled with pipets and large, expensive manipulators. Large quantities of biological medium including growth agents for human embryo culturing renders the corresponding in vitro procedure even more expensive. Livestock growth factors, for example, have costs exceeding $200 per 50 xcexcg.
Such static culture systems also fail to allow for changing the milieu in the culture medium as the embryo develops. Current culture systems with flowing medium have culture chambers as small as 0.2 to 0.5 ml. However, the culture volumes are greater than needed and medium is replenished too quickly. The endogenous growth factors that enhance development are diluted out and washed away. The large volumes of medium required substantially increase costs when expensive growth factors, such as IGF-II ($200 per 50 xcexcg) are used. In addition, known systems cannot track individual embryos.
Conventional handling techniques also provide limited ability to evaluate embryos. An ability to evaluate pre-implantation embryos, including embryos, pronuclear zygotes, and oocytes, would provide a better success rate for implantations. Currently, the morphology of most embryos is evaluated prior to their transfer into a recipient. Morphology examination will sort out embryos with gross defects but is not a highly reliable indicator of viability. Chemical monitoring over a period of time has been used, but requires numerous measurements over a period of time. The result is a much better predictor of viability, on the order of 80%, but many embryos fail to survive the monitoring process. This requires use of additional embryos. This results in multiple births, other complications, and entails additional labor costs.
Conventional techniques also provide harsh methods for removal of the zona pellucida, which is a critical step in the making of chimeric embryos. Conventionally, an embryo is mouth pipetted from one tissue culture dish containing the culture media, into a culture dish containing an acidic media. The embryo is left in the media for a period of time (tens of seconds) then mouth pipetted into a dish containing fresh culture media. The embryo is then flushed in and out of the pipette a few times to quickly disperse all of the acidic media and minimize damage to the cell membranes. The opening of the mouth pipette is about the same size as the embryo, and the flushing therefore causes sheer stresses on the embryos. The imprecision of mouth pipetting therefore provides ample opportunity for damage.
Thus, there is a need for an improved embryo handling device and method which addresses problems in known embryo handling techniques. An improved embryo handling device and method should provide for an improved simulation of natural conditions. It should also provide a building block upon which larger and/or more powerful and accurate instruments may be based, such as embryo culturing systems, embryo analysis systems, embryo storage systems, and similar systems. There is a further need for improved evaluation of embryo viability.
These needs are met or exceeded by the present microfluidic embryo handling device and method. The invention simulates biological rotating of embryos. An embryo fluidic channel moves an embryo inserted therein with fluid, and is sized on the same scale as the particular type of embryo or embryos to be handled. The sizing and fluid communication produces a simulated biological rotating of embryos. In addition, the fluid flow with and around the embryo or embryos prevents stagnation, reducing the likelihood of the embryo or embryos developing injuries that may be analogous to xe2x80x9cbed soresxe2x80x9d.
The invention also permits the biological medium fluid to be altered gradually, having significant advantages compared to repeatedly manually transferring an embryo from one medium to another medium in a pipet or petri dish. Gradual changes avoid the shock from sudden changes in local environment. The microfluidic system of the invention further permits the co-culturing of an embryo with other embryos, co-culturing of an embryo or embryos with cells upstream of the embryo(s), and maintenance of a separate control culture that shares a common biological medium with a subject embryo(s) thereby ensuring that test embryos see the same environmental conditions as the subject embryo(s).
Other aspects of the invention concern specific uses of the broader principles of microfluidic embryo handling to manipulate, evaluate and position embryos. One aspect concerns the use of a gradual series of constrictions to remove surrounding material from an embryo. This has been demonstrated to remove surrounding cumulus from oocyte. A first few constrictions cut the cumulus, which can then be sucked off of the oocyte in a final small constriction which is sized to prevent passage of the oocyte.
Embryo evaluation is also realized in accordance with the basic invention principles. In a preferred evaluation, surface properties and compliance (deformation) properties of embryos are evaluated. The microfluidic channels provide the opportunity for fine controls of pressure to conduct various evaluations at forces slightly below which damage to embryos is known to occur. Measurement of the distance and/or speed which embryos roll in a same pressure gradient microfluidic channel provides information, with healthy embryos traveling slower or a shorter distance as they demonstrate more stiction to channel walls. Positioned at a constriction, healthy embryos also appear to deform less than unhealthy embryos that are more readily pulled into a constriction. In addition, healthy embryos appear to resume their shape better.
Fluid from microfluidic channels is easily collected downstream without altering the embryo environment, providing a better opportunity for chemical analysis of fluid chemical analysis than convention manual handling and sampling techniques. In addition, all of the fluid collected from a microfluidic channel has passed over the embryo. This provides better evaluation information than fluids stagnant around an embryo in a Petri dish. Through the invention, medium can continuously or periodically pass over embryos and be collected downstream, eliminating additional handling required in a petri dish technique. The invention provides more consistent fluid samples since fluid can be repeatedly collected in the same manner, whereas samples taken from petri dishes may vary based upon placement of the pipette that suctions medium, i.e., how far or how close to an embryo.
Use of clear channel sections allows for many types of optical analysis. Stains or dyes may be added for visual inspection at clear sections. Clear sections also provide the opportunity to use image analysis devices, since the microfluidic channels may be configured to precisely position an embryo at a location for analysis by imaging equipment.
Precise position of embryos using channels and constrictions of the invention, and/or flow manipulation, further enables an improved method for zona pellucida removal of mammalian embryos. Embryos are moved through flow to a precise location where lysing agent can be washed over the embryo to achieve zona removal.